International Journal of Advanced Biotechnology and Research ISSN 0976-2612, Online ISSN 2278–599X , Vol 3, Issue 3, 2012, pp 635-643 http://www.bipublication.com

MODERATELY HALOPHILIC FROM SOLAR SALT PANS OF RIBANDER, GOA: A COMPARATIVE STUDY

1 2 3 Vaishali V Surve, Meena U Patil , Smita M Dharmadekari

1Department of Biotechnology, Vivekanand College Aurangabad, Maharashtra, India-431001 [email protected] 2Department of Zoology, Dr.BAMU, Aurangabad Maharashtra, India-431001 3Department of , Institute of Science, Aurangabad, Maharashtra, India-431001

[Received-08/07/2012, Accepted-01/08/2012]

ABSTRACT

Hypersaline environment of solar Salt pans are habitats of robust and diverse halophilic microorganisms .The present study makes an effort to isolate and compare the variation and species specific characteristic of the bacteria , isolated from the solar saltpans of Ribander, Goa.The moderate halophiles are studied considering their potential enzymatic applications. Comparative analysis yield valuable information of the extreme ecosystems .Besides, solar saltpans are endangered ecosystems, harbouring potential microbes. This study makes an effort to create awareness for conservation of this unique ecosystem. Solar saltpan water was used as a source for isolation after enrichment of the culture . Morphological , Biochemical, Antibiotic sensitivity, physiological and enzymatic(Protease )characteristics of the isolates were compared. The 16S rRNA sequencing and G+C analysis were used to identify and differentiate halophilic bacterial strains .The 2 moderately halophilic bacteria isolated were phylogenetically similar to Alkalibacillus HSD20 and Virgibacillus panthotheticus, speceis .The isolated bacteria displayed promising potential for protease production and varied species specific characters .The Ribander solar saltpans are a treasure of halophilic bacteria with a sparkling future. The study encourages further exploration of these extremophiles, their haloenzymes and saline habitats .

Key words: Solar salterns, Moderately halophilic bacteria, Protease, species specificity

[1]INTRODUCTION and salt marshes,or as artificial solar salterns Hypersaline environments are widely [1].Microorganisms that thrive in these distributed on the earth’s continent where they environments have been broadly classified exist either as natural water bodies such as into halophilic microorganism (that is, require permanent saline lakes, ephemeral saltpans salt for their viability) and halotolerant MODERATELY HALOPHILIC BACTERIA FROM SOLAR SALT PANS OF RIBANDER, GOA microorganisms which are able to grow in the to NaCl saturation. Characteristic salt-adapted absence as well as in the presence of NaCl. microbial communities are found along the Halophiles can be further divided into slight salinity gradient[6] . Solar saltpans are found halophiles that grow optimally in 3% (w/v) worldwide. Because of the increasing salinity total salt, moderate halophiles optimal growth of the environment, solar salterns are at 3 - 15% (w/v) salt and extreme halophiles considered extreme environments with very that grow optimally at 25% (w/v)salt [ 2 ]. restricted biology [7]. It is surmised that Halophilic and extremely halotolerant marine environments like solarsaltern may microorganisms are present in each of the yield newer strains which may prove to be a three domains of life: archaea, bacteria and rich resource of newer bioactive metabolites eukarya .The domain bacteria typically [8]. However, in spite of a growing interest in contains many types of halophilic and the use of halophilic enzymes for halotolerant microorganisms that spread over a biotechnological applications, there are large number of phylogenetic subgroups. Most relatively few reports in the literature about of these are moderate rather than extreme their production and characterization [9].As halophiles [3]. Scientific interest in industrial process conditions are harsh, there extremophilic microorganisms, especially are demands for biocatalysts that can hyperthermophiles, thermoacidophiles, withstand the process conditions. The majority archaebacterial anaerobes, and of the enzymes used to date originate from hyperhalophiles, has recently increased .One mesophilic organisms and, despite their many reason for this interest is the need to advantages, the application of these enzymes understand the biochemical mechanisms is restricted due to their limited stability at the involved under extreme conditions because of extremes oftemperature, pH and ionic possible biotechnological use of enzymes and strength. On the other hand, extremophiles are molecules from such organisms. Among a potent source of extremozymes,which show extremophilic bacteria, thermophiles are the outmost stability under extreme conditions. most intensively studied. In contrast, less Extremozymes have great economic potential attention has been paid to halophilic in many industrial processes( e.g. agriculture, microorganisms [4]. Halophilic and food, feed and drinks, detergents, halotolerant bacteria secrete a wide range of textile,leather, pulp and paper[10]. Out of the hydrolytic enzymes into their surrounding vast pool of Extremozymes, halophilic environment. Several of these enzymes which proteases are the most widely exploited include amylases, proteases, xylanases and enzymes in the processing of food, leather and cellulases display polyextremophilic detergents [11].Proteases are hydrolytic properties. They are generally haloalkaliphilic enzymes, which can degrade different protein and thermotolerant which renders them sources, so find potential application for waste amenable to an array of industrial processes, treatment, bioremediation, wool quality normally performed at extreme conditions of improvement, meat tenderization, in food, temperature and pH[ 5]. Multi-pond solar leather, pharmaceutical and detergent salterns, which are used worldwide for salt industries[12]. production along tropical and subtropical Goa’s traditional salt industry is said to have coastal areas, present an environment with been a major supplier of salt to the country increasing salt concentrations, from seawater and an exporter to some foreign countries

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MODERATELY HALOPHILIC BACTERIA FROM SOLAR SALT PANS OF RIBANDER, GOA since the 10 th century.Sparked by the area of this saltpan is 12,329.12m 2 (Figure 1 ) availability of microbial diversity data in these The site is subjected to heavy annual rains(125 salterns and the potentials of moderately cm) during the monsoons. The prevailing halophilic bacteria , an effort is made to study climate is characterized by three distinct and compare the species . Research aims at seasonsviz. Pre-monsoon: from February to making an effort to conserve these unique May; Monsoons: from June to September and microflora for biotechnology research and PostMonsoon: from October to January. The industrial use. climate on an average is generally warm and [II] MATERIALS AND METHODS humid,fluctuating from a minimum of 20 0C in 0 The traditional salt industry has been existing the month of December to 42 C in May.

in Goa since 500 A.D. Goan salt was Figure-1 Study area map: Shaded area depicts the considered to be of the best quality and was location of Ribander salterns exported to several African and Arabian countries during the post Medieval period. 2.2Sample Collection -Overlaying saltpan Goa has over 200 salt pans and produces water was collected in sterile glass bottles and o 35,000 metric tones of salt annually[13]. stored at 4 C. Samples were analysed within a 2.1Sampling site : The study site was the 24 hours of collection.The sample collection Ribandar saltpans (15 0 30.166 N and 73 0 was performed as specified in IS:162- 51.245 E) Goa, India and is situated along the 1981[14]. Mandovi estuary in Tiswadi taluka. The total

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2.3Enrichment and isolation of halophiles - and subsequently measuring their optical Halophiles were enriched in halophilic density[16].Salt concentration was maintained broth(Abrram gibbons media) containing at 15% and pH at 7.0. (gm/lit): Casein acid hydrolysate-5, Yeast 2.6 Antibiotic susceptibility test extract- 10, Proteasepeptone-5, Trisodium Halophilic bacteria are suseptable to citrate- 3, Potassiumchloride- 2, Magnesium antibiotics unlike the halophilic archaea . This sulfate- 20, Fe SO4-0.05 ,Sodiumchloride- character also differentiates the halophilic 150(15%), pH- 7.0 for 10-12 days [15]. From bacteria and archaea.Test for antibiotic enriched 15 % NaCl (w/v) halophilic broth, sensitivity was performed by the Kirby-Bauer cultures were streaked on respective agar disc method[16].The antibiotics tested were media by four sector method for the purpose Penicillin, Gentamycin,Streptomycin and of isolation into pure culture[16]. chloramphenicol.All in a concentration of 2.4Characterization of organisms -Gram 10mcg/disc(Himedia) staining ,Cell morphology and motility were 2.6 Genotypic analysis examined on freshly prepared wet mounts by A]16srRNA sequencing light microscopy. Colony pigmentation was The 16S rRNA genes were isolated from the recorded on agar plates after 10 days of cells PCR amplifications of the 16S rRNA growth was performed [16]. The cultures gene, from the purified genomic DNAs, were were purified on the same salt concentration carried out using the primer sets 27F (5´- and medium from which they were isolated. AGAGTTTGATCCTGGCTCAG-3´) and All tests were performed at 15 % salt 1492R (5´-GGTTACCTTGTTACGACTT- concentration . Cultures were tested for 3´).The purified PCR product was done using biochemical characteristics and carbohydrate BigDye Chemistry, and performed as per the utilization[17]. The isolates were identified manufacturer's protocols (Applied Biosystems according to [24,25]. 3730xl DNA Analyzer) The closest known 2.5 Physiological characterization relatives of the sequenced organisms were Salt concentration optimum - The isolates determined by sequence database searches. were screened for their salt tolerance level by The purified extension products were growing them on to nutrient broth tubes with separated in the ABI 3730xl DNA Analyzer concentrations of salt (NaCl) ranging from 0% by capillary electrophoresis. Sequence data to 25% and subsequently measuring their analysis was done using ChromasPro and optical density[16].pH was maintained at 7.0 Sequencing Analysis software. and temperature at 37 0C. B] G+C analysis: pH optimum - Nutrient broth was prepared at The mol%GC content of DNA should be part different pH range to detect the tolerance level of the description of the type strain of the type of the strains and subsequently measuring species of a new genus [18].1-2 g wet biomass their optical density at each pH [16].Salt (centrifugal pellet) are needed. The biomass concentration was maintained at 15% and can be sent either in frozen state on dry ice or temperature at 37 oC suspended in isopropanol/water (1:1, v/v) at Temperature optimum - All the isolates ambient temperature. Lyophilized cells in a were tested for the temperature tolerance by corresponding amount can also be sent. subjecting them to different temperature(10. , However, the use of lyophilized cells may 25 , 37, 40, 45, 50, 55, 60 o C) in nutrient broth result in a lower DNA yield.Cells are

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MODERATELY HALOPHILIC BACTERIA FROM SOLAR SALT PANS OF RIBANDER, GOA disrupted by French pressing and DNA is casein is known to lose its original purified on hydroxyapatit according to conformation at higher NaCl concentrations Cashion et al. [19]. The DNA is hydrolyzed [23]. One millilitre of casein buffer solution with P1 nuclease and the nucleotides are was preincubated at 37°C for 5 min.The dephosphorylized with bovine alkaline reaction was initiated by adding 1 ml of phosphatase (Mesbah et al., 1989). The enzyme. After incubation for 10 min at37°C, resulting deoxyribonucleosides are analyzed the reaction was terminated by adding 3 ml of by HPLC [20]. Lambda-DNA and three DNAs 5% (w/v) trichloroacetic acid(TCA). For blank with published genome sequences tubes TCA was added before enzyme. The representing a G+C range of 43-72 mol% are content was centrifuged and the absorbance of used as standards. G+C values are calculated supernatant was measured at 280 nm. One unit from the ratio of deoxyguanosine and of enzymeactivity was defined as 1 µg of thymidine according to the method of Mesbah tyrosine released per minute. et al. [21]. 2.7Screening of isolates for extracellular RESULT AND DISCUSSION enzyme production :Standard bacteriological Two halophilic species belonging to the tests for extracellular enzyme production were genus Alkalibacillus [A1] and Virgibacillus performed for protease enzyme . Cultures [V1] were isolated from the solar salterns of were spotted on skimmed milk-agar plates and Ribander . Both the cultures belonged to incubated at 37°C for 5-7 days. Presence of phylumXIII , class and zone of clearance surrounding the culture spot family . A1 belonged to the genus was taken as a measure of protease II. Alkalibacillus and V1 belonged to the production[22]. Genus XIX. Virgibacillus . The isolates Protease production by the isolates :The displayed varied species specific characters. isolates were further analysed for the amount The characteristic features of the 2 species of protease produced by them. The protease are compared in Table 1. The isolates production medium was similar to the growth displayed same morphology and presence of medium except that the yeast extract (10 g/l) spores. Yellow pigment production was seen was replaced by same quantity of soybean in A1 isolate, which appeared at salt meal. The isolates were grown in the concentration of 20% , however at 5%-15 % production medium at 37°Cfor 96 h under the colonies displayed creamish colour.This shaking conditions (200 rpm) and the enzyme characteristic is similar to the species activity was estimated at interval of 24 h. Alkalibacillus haloalkaliphilus .V1 isolate Enzyme activity displayed creamish grey colour.A1 was an The culture was centrifuged at 10,000 × g for alkaliphilic halophile ,with an pH optimum of 20 min (4ºC) and the culture supernatant was 9-10. Several biochemical tests were used as a source of protease. The caseinolytic performed to differentiate the isolates. activity was estimated using modified Anson’s Certain biochemical properties of method. The assay was performed at 37°C microorganisms also relate to their potential in using 1% casein as a substrate. The substrate Biotechnology. Both the isolates were was prepared in 50 mM Tris-HCl buffer (pH sensitive to the antibiotics(Table 1) ,a criterion 7.2) containing 2M NaCl. The concentration to differentiate the bacteria from archaea. The of NaCl was set at 2 M in the assay system as physiological characterization revealed

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diverse values for both the species.The 8.0[28]. Similarly a Gram-positive, strictly bacteria isolated had an optimum growth aerobic and moderately halophilic bacterial value in the range of 5-15% NaCl, and thus strain was obtained from the sea water can be classified as moderately halophilic sample collected from International Sea Water bacteria accoding to Ventosa et al 1998[2]. Bathing Place in Weihai, a city on the shore of Fritze [2002] recommended that phenotypic the Yellow Sea. [29] Alkalibacillus characterization results should not be directly filiformis sp.was., isolated from a mineral pool compared without full background knowledge in Campania, Italy[31].Recently Virgibacillus of the precise conditions used for a particular albus sp. nov., a novel moderately halophilic test. This can be particularly true for the group bacterium isolated from Lop Nur salt lake in of Gram-positive endospore-forming bacteria Xinjiang province, China.[32] Virgibacillus that were formerly classified as the genus salarius sp. nov., a halophilic bacterium Bacillus but have now been reclassified based isolated from a Saharan salt lake in 2008[33], upon phylogenetic diversity into separate and shared similarity with our isolate V1. lineages [26].Therefore, we also used 16S Screening of isolates for extracellular rDNA sequence analysis to ensure the protease production- accurate taxonomic position of the halophilic strains reported in this study[Table 2]. G+C Most halophilic bacteria are known to secrete analysis also revealed the diversity and proteases into the external environment that ultimately the specificity of species at the can have unique properties for application to genotypic level. On the basis of the biotechnology. A1 showed significant zone of phenotypic characteristics and the comparison clearance , an indication of protease of partial 16S rRNA gene sequences, the production. V1 showed comparatively less isolates were identified, and were zone of clearance on skimmed milk agar phylogenetically similar to Alkalibacillus plates. These results were indicative HS20D [A1] and Virgibacillus Panthotheticus of Alkalibacillus species as potential protease [V1], which was well supported by the producers. The results are supported byFrizte phenotypic, biochemical, and physiological 1996[30]. Hence can be investigated for characteristics. Alkaliphilic micro-organisms, further quantification of protease mainly those belonging to the genus Bacillus , production.Protease production by these are of considerable industrial interest, organisms in soybean meal-based production particularly for the production of enzymes medium was studied as a function of time (proteases, xylanases, Maximum protease production 14.24 ± 1.55 glycosidases,Alkaliphilic micro-organisms are U/ml was observed from A1 at 72 h. Protease also often halophilic. There are a large number production by V1 was 12.76 ± 0.09 U/ml of recognized species of the which was comparatively less and was also genus Bacillus and related genera that could achieved at 72 h of growth. These results were be described as moderately halophilic or suggestive of A1 as potential isolate for halotolerant, some of which are also production of protease. alkaliphilic [30]. Alkaliphilic halophilic, Gram- positive, spore-forming motile Bacillus-like strain CONCLUSION YIM 012, was isolated from one of the hypersaline Comparative studies of micro flora in the soil samples collected in Xin-jiang province, extreme environments results in better China. Its optimum growth occurred at pH 7.0-

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.Process Biochem . 38: 571-579. Table 1- Morphological, Biochemical, Enzymatic and [24]De la haba RR, Sanchez Parro C, Marquez M genotypic characteristics of the two halophilic species . C, Ventosa A.[2011] of Halophiles Tests A1 V1 Horikoshi koki(Ed).Extremophiles Handbook Gram’s Stain Positive Positive .Springer NY, Antranikian Garo,Bull A T, Robb Endospore staining + + FT,Stetter K O(Co-Ed). [25] ]Bergeys D H, Ioone D R. [2009] Bergeys Cell shape Long Rods Rod Pigments Yellow Creamish manual of systematic bacteriology2 edition Vol 3 grey The Firmicutes. Springer Newyork Lanyi. H2S Production - +[weak] [26] Stackebrandt E, J Swiderski. [2002] From Nitrate reduction _ + phylogeny tosystematics: the dissection of the Motility + + genus Bacillus. In: Applications and Systematics of Catalase + + Oxidase - + Bacillus and Relatives ,(Eds.): R. Berkeley, M. NaCl optimum 10 -15% 5- 10% Heyndrickx, N. Logan and P. DeVos. pp. 8-22. pH optimum 8-9.0 7.0 Oxford: Blackwell o o [27] Fritze D [2002]. Bacillus identification- Temperature optimum 30-37 C 30-37 C traditional approaches.In: Applications and Systematics of Bacillus and Relativ es ,(Eds.): R. L-Arabinose _ + D-Mannitol _ -

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D-Lactose _ + D-Trehalose _ + D-Glucose + + D-Galactose _ _ D-Fructose _ + G+C 38 % 37% Antibioticsensitivity(Penicil s s lin, Gentamycin,Strptomycin and chloramphenicol.)

Key-+[positive results/acid formation] _[Negative results] S[Sensitive]

Table 2. Identification of isolated halophilic strains based on 16S rRNA gene sequence and their accession numbers (BLAST similarity search results)

Number of Accession Sequence Strain Strain name / Closely related nucleotides of number of 16S similarity (%) of ID Genus taxa 16S rRNA gene rRNA gene 16S rRNA gene Alkalibacillus sp A1 Alkalibacillus sp 1463 EF517966.1 98% HS20D

Virgibacillus V1 Virgibacillus sp 1533 JN791392.1 88% pantothenticus

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